In real-time PCR, quantitative information is obtained from the cycle threshold (Co), a point where the fluorescence curve signal increases above background. External calibrators are required to estimate the concentration of unknown samples. The variation in amplification efficiency affects Cq values which in turn limits the accuracy of techniques for absolute quantitation. It is recognized that the combination of limiting dilution, end-point PCR, and Poisson statistics can determine the absolute measure of nucleic acid concentration (Vogelstein and Kinzler, U.S. Pat. No. 6,440,706).
Digital single-nucleotide polymorphism (SNP) analysis has been developed to amplify a single template from a pool of DNA samples, thereby generating amplicons that are homogeneous in sequence. Different fluorescently-labeled probes are used to detect and discriminate different alleles (e.g. paternal vs. maternal alleles or wild-type vs. mutant alleles), which can be readily counted. The advantages of digital PCR have been reported (Chang and Shih). Currently there are basically two commercially available digital PCR systems. The first one uses microfluidic chambers or micro-wells to split samples into aliquots on the order of hundreds of nanoliters. The second system involves emulsion PCR, wherein templates are clonally amplified in the presence of beads which are recovered from the emulsion, then hybridized with a hybridization probe and read by conventional flow-cytometry. These approaches are ideally suited for single-nucleotide polymorphism analysis and for allelic imbalance detection (Hindson, Tsui; the content of which is incorporated herein by reference). Different probes have been used for the detection of digital PCR-generated amplicons, for example, molecular beacons (U.S. Pat. No. 6,440,706) and MGB TaqMan and dual-labeled non-MGB TaqMan probes (Hindson). Typically, these probes are longer than 16 bases. The TaqMan probes are cleaved by polymerases with 5′-exonuclease activity. In some applications droplet digital PCR (ddPCR) is used to make highly precise measurement of DNA, for example HIV DNA (Strain).
Digital PCR is performed to end-point (35-45 cycles) in a conventional thermocycler. Samples and oil are introduced into a droplet generator and loaded into 96-well PCR plate. Droplets from each well are placed onto a reader which moves them in single file past a two-color fluorescent detector at room temperature. Although digital PCR shows vast improvement in precision and sensitivity, longer probes tend to hybridize non-specifically.
Minor groove binder-fluororophore-oligonucleotide-quencher (MGB-Fl-oligo-Q) probes have been disclosed in U.S. Pat. Nos. 7,381,818 and 7,759,126, the content of which is incorporated by reference, but no detection of ddPCR-generated amplicons at room temperature with these short probes has been disclosed.